Proper functioning of the brain depends on the coordination of diverse types of neurons and glia, which arise at distinct locations and make intricate connections. Understanding how these brain cells are normally developed holds the key to therapeutic intervention of neurological diseases, especially those with developmental etiology. Neural stem cells (NSCs) play critical roles in generating neurons and gila during both embryonic development and adult life. Despite their clinical importance, little is known about the identity and location of the mammalian NSCs and how their behavior is regulated in vivo. The Drosophila NSCs provide an excellent opportunity for such studies. The long-term goal of this proposal is to understand the molecular and cellular mechanisms regulating the differentiation and self-renewal of NSCs, using cell biological, genetic, and molecular analyses in Drosophila. The Drosophila central nervous system stem cells (neuroblasts) undergo self-renewing asymmetric cell divisions. After each division, one daughter cell remains as a stem cell and the other is committed to differentiation. Determinants whose asymmetric segregation serves to intrinsically alter cell fate choices have been identified. The asymmetric segregation of determinant Numb requires Partner of Numb (Pon) protein. In this proposal, we plan to use Pon as a starting point to achieve a mechanistic understanding of how the asymmetric segregation of cell fate determinants is accomplished. We will first carry out a detailed characterization of the Pon localization domain to identify the molecular features in this domain that contribute to its asymmetric localization and proteins that act through this domain to exert their function. To identify other players that control the localization of Pon, we will test the involvement of myosin motor molecules. We will also perform a forward mutagenesis screen, using a Pon-green fluorescent protein (GFP) reporter as the assay system, to systematically identify key genes that control the asymmetric division of NSCs. Given the conservation of molecular mechanisms that control many developmental processes, it is highly likely that the genes and genetic programs regulating the behavior of NSCs will also be conserved between Drosophila and mammals. We believe that knowledge gained from our study will influence mammalian NSC research and contribute to the understanding and treatment of neurological diseases in humans.